There is increasing recognition that immunotherapy is a promising approach to treat cancer. Various strategies have been proposed including treatment with cytokines such as interleukin-2 (IL-2). IL-2 impacts various immune cell types including T and B cells, monocytes, macrophages, lymphokine activated killer cells (LAK) and NK cells [10, 40].
There have been proposals to concentrate cytokines at the site of tumors to help increase efficacy. Typical methods include direct injection of the cytokine or gene encoding same into the tumor, or targeted delivery of the cytokine by fusing it to a tumor antigen specific antibody [20]. However, these methods have drawbacks.
For example, most direct injection methods are difficult to use especially at early stages of cancer when tumors are typically small (micrometastases). Moreover, such methods are usually labor-intensive with little guarantee of therapeutic success. This makes treatment of large patient populations impractical and costly.
Antibody-cytokine fusion constructs have been used in an approach to treat cancer. However, the methods are limited to the extent that the antibody has a limited binding spectrum. That is, the antibodies can only recognize certain cell surface antigens. Unfortunately, many tumor antigens are not displayed appropriately for antibody recognition, thereby limiting potential of antibody based approaches. Moreover, there are reports that many tumor specific antigens are derived from aberrant expression of cell type specific proteins. These may exist only with a small number of tumor types. This drawback limits the potential of antibody based therapies even further.
The p53 protein is an intracellular tumor suppressor that has been reported to act by arresting abnormal cells at the G1/S phase of the cell cycle. Over expression of the protein is believed to be a significant tumor marker for a large number of human malignancies and there is recognition that it is a good target for broad spectrum targeted tumor immunotherapy. The p53 protein is usually displayed on the cell surface in the context of major histocompatibility complex proteins (MHC). Such protein complexes are known to be the binding targets of T-cell receptors (TCRs). [49].
There have been attempts to use certain TCRs to detect MHC/peptide complexes containing peptide (Epel et al., 2002; Holler et al., 2003; Lebowitz et al., 1999; Plaksin et al., 1997; Wataya et al., 2001; O'Herron et al., 1997). However, these and related methods have significant shortcomings.
For instance, many of the methods require that TCR constructs be multimerized (i.e., designed to have multiple TCR copies) presumably to enhance peptide antigen binding with peptide antigen artificially. Target (antigen presenting) cells are often manipulated by the methods to express relatively large amounts of peptide antigen. Sometimes the density of peptide antigen is as high as 104 to 105 complexes per cell (Wataya et al., 2001). Such a high peptide antigen density is believed to facilitate binding and detection by the TCRs. However, these levels of peptide antigen are artificial and typically much greater than the level of MHC/peptide complexes that include most tumor-associated antigens (TAAs). For some TAAs, less than about 50 HLA/peptide complexes per cell are present (Pascolo et al., 2001; Schirle et al., 2000). Thus, there has been recognition that the prior methods are not sensitive enough to detect most if not all TAAs.
There have been attempts to use certain TCRs to detect cells expressing particular peptide antigens. Like many antibody based methods, these approaches have either lacked enough sensitivity to detect peptide antigen or failed to detect such antigen completely.
For example, Holler et al. (2003) reported the development of certain soluble TCRs that were reported to react with MHC/peptide complexes. Although the TCRs were able to detect antigen with cells artificially “loaded” with the antigen, the molecules were unable to detect endogenous antigen on tumor cells. Holler et al. concluded that when the antigen is present at a density of less than 600 copies per cell, TCR based methods are not sensitive or reliable enough to detect antigen.
Particular TCR based methods have been used to detect viral peptides in the context of MHC molecules. (Strominger, et al., WO9618105). However, these and related methods suffer from drawbacks. For instance, there is general recognition that viral infection often produces exceptionally high densities of MHC/peptide complexes, typically approaching from >1000 to >105 complexes per cell. See Herberts et al., 2001; van Els et al., 2000. Thus like most other peptide antigen detection methods, TCR based approaches to detect viral antigens have so far relied on the relatively large number of antigen targets to drive the detection method.
Although some TCR based methods have been used to detect relatively large amounts of peptide antigen, it is less certain if the methods will work when the TCR is fused to other molecules such as a cytokine, an immunoglobin domain such as IgG1, biotin or streptavidin. That is, it is not certain how the resulting fusion molecule will impact the TCR peptide binding groove particularly when low densities of TAA need to be analyzed. Small distortions in the TCR peptide binding groove, while not necessarily problematic when relatively large amounts of peptide antigen are to be analyzed, could reduce TAA binding specificity and selectivity. Even small changes in the TCR peptide binding groove function could jeopardize detection of cancer cells that express low TAA densities.
It would be useful to have methods for detecting TAAs that are sensitive, selective and reproducible especially when the peptide antigens are present in low densities. It would be especially useful if such methods could be used with a variety of soluble TCRs including molecules such as those fused to a detectable label or a cytokine.